Genome-based classification of Acidihalobacter prosperus F5 (=DSM 105917=JCM 32255) as Acidihalobacter yilgarnensis sp. nov

Abstract

The genus Acidihalobacter has three validated species, Acidihalobacter ferrooxydans, Acidihalobacter prosperus and Acidihalobacter aeolinanus, all of which were isolated from Vulcano island, Italy. They are obligately chemolithotrophic, aerobic, acidophilic and halophilic in nature and use either ferrous iron or reduced sulphur as electron donors. Recently, a novel strain was isolated from an acidic, saline drain in the Yilgarn region of Western Australia. Strain F5^T has an absolute requirement for sodium chloride (>5 mM) and is osmophilic, growing in elevated concentrations (>1 M) of magnesium sulphate. A defining feature of its physiology is its ability to catalyse the oxidative dissolution of the most abundant copper mineral, chalcopyrite, suggesting a potential role in biomining. Originally categorized as a strain of A. prosperus, 16S rRNA gene phylogeny and multiprotein phylogenies derived from clusters of orthologous proteins (COGS) of ribosomal protein families and universal protein families unambiguously demonstrate that strain F5^T forms a well-supported separate branch as a sister clade to A. prosperus and is clearly distinguishable from A. ferrooxydans DSM 14175^T and A. aeolinanus DSM14174^T. Results of comparisons between strain F5^T and the other Acidihalobacter species, using genome-based average nucleotide identity, average amino acid identity, correlation indices of tetra-nucleotide signatures (Tetra) and genome-to-genome distance (digital DNA-DNA hybridization), support the contention that strain F5^T represents a novel species of the genus Acidihalobacter. It is proposed that strain F5^T should be formally reclassified as Acidihalobacter yilgarnenesis F5^T (=DSM 105917^T=JCM 32255^T).

Keywords: Acidihalobacter; Yilgarn Craton; acidophile; average amino acid identity (AAI); chalcopyrite bioleaching; genome-based average nucleotide identity (ANI); genome-to-genome distance (digital DNA-DNA hybridization (dDDH); halotolerant; iron and sulfur oxidising.

Fig. 1.

Electron microscopy image of strain F5^T grown in the presence of 214 mM NaCl. The scale bar is 200 nm.

Fig. 2.

Maximum-likelihood phylogenetic tree of 16S rRNA gene sequences of strain F5^T (in red) and other phylogenetic relatives as described in the text. Bootstrap percentages (1000 replicates) are labelled at the nodes. Scale bar represents 0.03 nucleotide substitution per site. The genetic distance of Halothiobacillus neapolitanus ATCC 23641 is not to scale as indicated by the break lines //. The full list of NCBI accession numbers is given in Table S1.

Fig. 3.

Phylogenomic trees of 14 members of the order Chromatiales and Halothiobacillus neapolitanus ATCC 23641 as outgroup, including strain F5^T (in red), based on (a) 30 concatenated conserved proteins from proposed 34 ribosomal proteins [30, 31] and (b) nine concatenated housekeeping genes. Statistically supported bootstrap values as percentages of 1000 replicates are labelled at the nodes. Scale bar represents 0.07 amino acid and 0.2 nucleotide changes per site, respectively. The full list of COG families is given in Table S2.

Fig. 4.

Heat maps of the percent difference of 16S rRNA gene sequences between the three validated Acidihalobacter species and strain F5^T. The results are displayed as a cladogram based on the 16S rRNA gene phylogenetic tree shown in Fig. 2, using 97, 98.7 and 98.73 % 16S rRNA gene sequence similarity cutoff values (left to right, respectively).

Fig. 5.

Heat maps indicating the genetic relatedness between the three validated Acidihalobacter species and strain F5^T displayed as a cladogram using different non-sequence-based methods. (a) dDDH, digital DNA–DNA hybridization, (b) ANI, average nucleotide identity, (c) AAI, average amino acid identity and (d) Tetra, nucleotide signature correlation index. The cutoffs for each of the four methods represent the values accepted as defining different species.